DNA structure and interactions with proteins are universally important in molecular biology. The extrachromosomal DNAs are often covalently closed circular and supercoiled. In addition to containing genetic information of their own, these structural forms are generally the mediators of integration and recombination. The expression of these functions involves interaction with proteins, both structural and enzymatic. OBJECTIVES: We plan to characterize the mechanism of selected DNA-protein interactions in two biological systems. The drug resistance factors (R-factors) of prokaryotics have been chosen because (1) they are of great medical importance; (2) they are readily isolated as closed duplex DNAs; and (3) they exhibit a wide range of intramolecular recombination phenomena giving rise to independent closed duplex submolecules. Vaccinia virus has been chosen as a second system because (1) again, the poxviruses (of which it is typical) are of health-related significance; (2) it contains a large number of enzymes in the virion, allowing the study of replication, transcription and translation in a relatively simple environment; (3) the genome, although fairly large, permits genetic analysis and the selection of specific mutants; and (4) discrete, biologically active nucleoprotein complexes can be isolated and examined. METHODS: We will combine biological (in vivo and in vitro) and chemical techniques. The DNA structures will be determined using restriction enzyme digestion, gel electrophoresis, electron microscopy and sedimentation. Protein-DNA complexes will be mapped using psoralen, a chemical crosslinking reagent. Temperature-sensitive mutants of vaccinia will be obtained. The mechanism of action of the vaccinia DNA topoisomerase will be determined, and a search for a gyrase will be initiated. Covalent DNA-protein complexes will be detected, and strong protein binding sites will be mapped by electron microscopy and restriction enzyme analysis.